Endothelium-derived nitric oxide (NO) is a critical mediator of vascular homeostasis. In normal vessels an increase in cardiac output potently increases regional tissue perfusion via flow- induced activation of endothelial nitric oxide synthase (eNOS, also known as type III NOS). The latter oxidizes L-arginine to citrulline and NO, the major endothelium-derived relaxation factor. In addition to its dynamic regulation of vessel tone, NO has become increasingly recognized for its long-term role in maintaining normal vessel structure. Because eNOS/NO function is dysregulated in major cardiovascular diseases, increasing our understanding of eNOS regulation in health and disease is of critical importance. We present exciting new data including the unequivocal determination of three phosphorylated eNOS amino acid residues, which constitute the first phosphorylation sites to be identified in NOS. We now propose to extend our previous work on the mechanotransduction of fluid shear stress (FSS) to NO production, given the importance of laminar FSS in the homeostatic functions of endothelium, via the following Specific Aims: Aim 1.To identify the amino acid sites of eNOS phosphorylated in endothelial cells. Aim 2. To characterize the regulation of eNOS function by phosphorylation of serine residues 634, 1179 and 116, comprising sites designated S1, F1 and F2. Aim 3. To determine the mechanotransducers of fluid shear stress to eNOS phosphorylation. Aim 4. To elucidate the regulatory role of eNOS phosphorylation in regenerating endothelial cells in culture and in denuded rat aorta.